Possible role of the Nt-4/1 protein in macromolecular transport in vascular tissue

Abstract

The Arabidopsis thaliana 4/1 (At-4/1) protein has a highly α-helical structure with potential to interact both with itself and other protein ligands, including the movement proteins of some plant viruses; the Nicotiana tabacum ortholog (Nt-4/1) has similar structure. Here we describe localization of GUS expression in transgenic N. tabacum seedlings under control of the Nt-4/1 promoter, which indicates that transcription is associated with the veins at certain developmental stages, and especially in the hypocotyl. Viroid accumulation and movement was altered in plants in which 4/1 expression was reduced by virus-induced gene silencing. These localization studies support a role of 4/1 in signaling in the vasculature,including mobility of pathogen-related and cellular RNAs.

Figure 1. Tissue-specific activity of the Nt-4/1 promoter at different developmental stages. Promoter activity was analyzed in transgenic Nicotiana tabacum cv Samsun nn plants carrying the GUS gene under the transcriptional control of a DNA fragment comprising 2000 base pairs upstream of the Nt-4/1 gene transcription start site experimentally mapped earlier. This region, presumably containing the Nt-4/1 promoter, was cloned from the “long” Nt-4/1 gene, which is expressed 3 times more efficiently than the “short” Nt-4/1 gene. The resulting construct was transformed into N. tabacum as described earlier. Detection of GUS was performed according to Jefferson et al. (A) Seedling with 2 expanded cotyledons and emerging foliage leaves. (B) 2-leaf stage. (C) 8-leaf stage. At this stage, staining was found in the petioles of leaves 1–3 but not in the cotyledons, and was also associated with the veins of leaf 4. In leaf 5 weak staining was found in the petiole and larger veins; in leaf 6 only faint staining of the midrib was observed; and no staining was found in the youngest leaves. At this developmental stage the hypocotyl stained very intensively, whereas the rest of the stem exhibited much weaker staining. (D–F) Anatomy of stained leaves in transverse section. A leaf which exhibited pronounced vein staining was taken from a 6-leaf plant for sectioning. For light microscope observations, material was fixed in 70% ethanol, dehydrated, embedded and then sectioned with a rotary microtome using standard methods of Paraplast embedding and serial sectioning at 10–16 µm thickness. Sections were stained with safranin and mounted in DPX mounting medium. (D) Thick vein with well-developed secondary xylem whose cells are arranged in regular radial rows. Arrowheads point to adaxial phloem. (E and F) minor veins composed exclusively of primary xylem and phloem. Xy, xylem; Ph, phloem; P, parenchyma cells.

Figure 2. Tissue-specific inactivation of the Nt-4/1 mRNA. The GFP-fusion of Nt-4/1 protein described previously was transiently expressed in young N. benthamiana leaves by agroinfiltration. Fluorescence was observed by confocal laser scanning microscopy in detached leaves 3 d after infiltration. (A) Nt-4/1-GFP expression in the presence of transiently co-expressed tombusvirus silencing suppressor p19; (B and C) Nt-4/1-GFP expression in the absence of p19. Scale bars, 50 µm in (A and B) 25 µm in (C).

Figure 3. Dot-blot detection of PSTVd in leaves of N. benthamiana plants. Two leaves from the young N.benthamiana plants were inoculated with AltMV.4/1 or control AltMV vector constructs. Quantitative reverse transcription (qRT-PCR) was performed to assess the relative 4/1 mRNA levels using an endogenous gene (actin) as an internal standard. At 13 dpi 4/1 expression was found to be strongly (approximately 8-fold) downregulated in systemic leaves of plants inoculated with the AltMV.4/1 construct (data not shown). At this time, the uppermost 1.5–2 cm leaf of both AltMV.4/1-infected and AltMV-infected control plants was challenge inoculated with infectious PSTVd RNA transcripts. Samples (1 disk per leaf) were collected from plants infected with each construct 9 and 21 d after inoculation with PSTVd (inoculated leaf) and 21 d (2nd–12th leaves above inoculated) for dot blot analysis using chemiluminescent detection with digoxygenin-labeled cPSTVd RNA probe and CDP-Star substrate. Note that each spot represents a pooled sample contained discs from 5 individual plants in that treatment. For systemic leaves numbers indicate the position of leaves above the challenge-inoculated leaf. “+ control”, PSTVd RNA transcripts used as a control.